How to get dose depedent result from luciferase assay? - (Mar/12/2006 )

Hi, everyone: I am doing luciferase assay by using NFkB as reporter and b-Gal to normalize the transfection effeciency. The experiment design is this: NFkB+b-gal+Plasmid A(0.25ug)NFkB+b-gal+Plasmid A(0.25ug)+plasmidB(0.25ug)NFkB+b-gal+Plasmid A(0.25ug)+plasmidB(0.5)NFkB+b-gal+Plasmid A(0.25ug)+plasmidB(1.0) Plasmid A encondes a protein that is well-known to activate NF-kB,so the purpose of my experiment is to see how the protein econded by plasmid B affect the protein encoded by plasmid A. my hypothesis is B positivelly affect A to activate NFkB,which has been demonstrate by western and now I want to use luciferase to confirm it further. However I met some problomes:1.I can not get dose-dependent effect. when I use 0.25ug B, it synergize the A effect to activate NFkB, but when I use higher amonut of B, it has the same effect as that when I use 0.25ug B or even reverse the effect( I mean the RLU even lower than that of 0.25B)

2. The more strange is the b-gal assay. for the control group(NFkB+b-gal+Plasmid A(0.25ug)), I can not get b-gal reading, but for all the other group I got good consistant reading. This definitely ruin my whole exp.I have used PCD plasmid to equal the amount of plasmids I transfect into every well so I can not figure our how this could happen. Can anyone help me analysis the problems?? I would appreciate any help!

-popogirlxd-

The problem with all those "normalizers" is that in many cases their promoter is actually responsive to the treatment/protein being tested. According to your description this might be the case. What is the promoter upstream of the b-gal gene? It might be responsive to protein B.about the increasing plasimd amounts, do you complete the amount of plasmid to the same total amount using an empty vector? this is important and failure to do so might reduce transfection efficiency.